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Applied Biochemistry and Microbiology (v.46, #6)
The effect of AmtR on growth and amino acids production in Corynebacterium glutamicum by N. Hao; M. Yan; H. Zhou; H. M. Liu; P. Cai; P. K. Ouyang (pp. 561-566).
AmtR, the master regulator of nitrogen control in Corynebacterium glutamicum, plays important roles in nitrogen metabolism. To investigate the influence of AmtR on amino acids production in C. glutamicum ATCC 13032, the amtR deletion strain C. glutamicum Q1 was constructed and cultured in modified CGXII minimal medium for 60 h. The ammonium consumption rates as well as amino acids production of both strains cultured in modified CGXII minimal medium were determined. The amtR deletion in C. glutamicum caused an obvious growth defect in the exponential growth phase, but both strains had the same biomass in the stationary phases. Maybe the less α-oxoglutarate was used for the tricarboxylic acid cycle to influence the growth of strains. During 12 h, the rate of ammonium consumption and the concentration of Glu, Pro, Arg and Ser were higher but Asp, Gly, He, Leu, Lys were lower in the mutation strain. During 48 h, the Q1 had higher levels of Asp, Lys, Pro, Ala and Val, and lower levels of Glu, Arg, Leu and Ile, compared to the wild. The more Glu was synthesized by the activated GS/GOGAT pathway in Q1, and then the accumulation of relative amino acids (Pro, Arg and Ser) were up-regulated within 12 h growth. After 48 h growth, the amtR deletion obviously influenced accumulation of Ala, Asp and Pro. The amtR deletion could influence the growth and amino acids production, which could be useful to the production of amino acids.
Protective action of reactivating factor of Luteococcus japonicus subsp. casei toward cells of Escherichia coli reparation mutants inactivated with UV-light by L. I. Vorob’yova; A. V. Fedotova; E. Yu. Khodzhaev (pp. 567-573).
Reactivating factor (RF) from Luteococcus japonicus subsp. casei had a protective action on UV-irradiated cells of Escherichia coli AB1157 with a native reparation system and on cells of isogenic reparation mutants of E. coli UvrA−, RecA−, and PolA−: the effect resulted in multifold increase of survivability. Defense action of L. casei exometabolite is not connected with stimulating reparation systems in E. coli, and, probably, it is mediated by involvement of the exometabolite in the mechanism of cell division. RF did not provoke the reactivation of E. coli cells inactivated by UV-light.
Characteristics of proteins synthesized by hydrogen-oxidizing microorganisms by T. G. Volova; V. A. Barashkov (pp. 574-579).
The study was conducted to determine the biological value of proteins synthesized by hydrogen-oxidizing microorganisms—the hydrogen bacteria Alcaligenes eutrophus Z1 and Ralstonia eutropha B5786 and the CO-resistant strain of carboxydobacterium Seliberia carboxydohydrogena Z1062. Based on a number of significant parameters characterizing the biological value of a product, the proteins of hydrogen-oxidizing microorganisms have been found to occupy an intermediate position between traditional animal and plant proteins. The high total protein in biomass of these microorganisms, their complete amino acid content, and availability to proteolytic enzymes allow for us to consider these microorganisms as potential protein producers.
Extracellular proteolytic activity of bacteria from soda-salt lakes of Transbaikalia by E. V. Lavrenteva; Ya. E. Dunaevsky; L. P. Kozyreva; A. A. Radnagurueva; B. B. Namsaraev (pp. 580-585).
Influence of nitrogen source on proteinases synthesis in aerobic alkalotolerant and halotolerant bacteria from soda-salt lakes of Transbaikalia was studied. Maximal accumulation of proteinases was revealed on medium with peptones. Introduction of various sources of nitrogen in the medium did not result in increase of enzyme activity in cultural liquid. It was indicated that secreting proteinases of the studied bacteria strains possess narrow substrate specificity, hydrolyze proteins and n-nitroanilide substrates have maximal activity during GlpAALpNA hydrolysis. Data of inhibitory analysis and substrate specificity of studied extracellular enzymes indicate that they belong to a class of serine proteinases of subtilisin-like type.
Oxidative biotransformation of thioanisole by Rhodococcus rhodochrous IEGM 66 cells by A. A. El’kin; V. V. Grishko; I. B. Ivshina (pp. 586-591).
Comparative study of sulfoxidation activity of free and immobilized Rhodococcus rhodochrous IEGM 66 cells was performed. Free Rhodococcus cells (in the presence of 0.1 vol % n-hexadecane) displayed maximal oxidative activity towards thioanisole (0.5 g/l), a prochiral organic sulfide, added after 48-h cultivation of bacterial cells. Higher sulfide concentrations inhibited sulfoxidation activity of Rhodococcus. Use of immobilized cells allowed the 2-day preparatory stage to be omitted and a complete thioanisole bioconversion to be achieved in 24 h in the case that biocatalyst and 0.5 g/l thioanisole were added simultaneously. The biocatalyst immobilized on gel provides for complete thioanisole transformation into (S)-thioanisole sulfoxide (optical purity of 82.1%) at high (1.0–1.5 g/l) concentrations of sulfide substrate.
Degradation of chlorinated biphenyls and products of their bioconversion by Rhodococcus sp. B7a strain by D. O. Egorova; E. S. Shumkova; V. A. Demakov; E. G. Plotnikova (pp. 592-598).
Strain Rhodococcus sp. B7a isolated from artificially polluted soil destructs mono- and di-substituted ortho- and/or para-chlorinated biphenyls with utilization of chlorinated benzoic acids and shows high degradation activity as regards trichlorinated biphenyls. It is shown that p-hydroxybenzoic and protocatehoic acids are the products of p-chlorobenzoic acid catabolism.
Intensification of surfactant synthesis in Rhodococcus erythropolis EK-1 cultivated on hexadecane by T. P. Pirog; T. A. Shevchuk; Yu. A. Klimenko (pp. 599-606).
Activity of key enzymes of n-alkane metabolism was determined in cells of Rhodococcus erythropolis EK-1, a surfactant producer grown on n-hexadecane. Potassium cations were found to inhibit alkane hydroxylase and NADP+-dependent aldehyde dehydrogenase, while sodium cations were found to activate these enzymes. Decreased potassium concentration (to 1 mM), increased sodium concentration (to 35 mM), and addition of 36 μmol/l Fe(II), required for alkane hydroxylase activity, resulted in increased activity of the enzymes of n-hexadecane metabolism and in a fourfold increase of surfactant synthesis. A 1.5–1.7-fold increase in surfactant concentration after addition of 0.2% fumarate (gluconeogenesis precursor) and 0.1% citrate (lipid synthesis regulator) to the medium with n-hexadecane results from enhanced synthesis of trehalose mycolates, as evidenced by a 3–5-fold increase in phosphoenolpyruvate synthetase and trehalose phosphate synthase, respectively.
Composite biodegradable materials based on polyhydroxyalkanoate by I. N. Gogotov; V. A. Gerasin; Ya. V. Knyazev; E. M. Antipov; S. Kh. Barazov (pp. 607-613).
Conditions for the processing and mixing of biodegradable polymers at temperatures less than their thermal destruction (130–150°C) using standard equipment have been identified. The structure of the polyhydroxybutyrate/valerate (PHB/V) copolymer has been revealed and peculiarities of the crystal phase formation at different monomer ratios have been investigated. It was shown that pure PHB with molecular mass 180–270 kDa has elastic module approximately 1.2 GPa, strength approximately 25 MPa, and elongation at break approximately 10%. The most active biodestructors of PHB, PHB/V, and their composites have been selected (Aspergillus caespitosus), and the ability of basidiomycete Panus tigrinus to biodegrade polyalkanoates was demonstrated for the first time. It was shown that A. caespitosus degraded PHB/V and Biopol films along with the PHB with the destruction rate depending on the technology of the film production, on the molecular mass, and on the extend of the polymer crystallinity.
Structure peculiarities of cell walls of Acremonium chrysogenum—an autotroph of cephalosporin C by T. S. Kalebina; I. O. Selyakh; A. A. Gorkovskii; E. E. Bezsonov; M. A. El’darov; M. I. Novak; A. G. Domracheva; Yu. E. Bartoshevich (pp. 614-619).
Alterations of cell walls of Acremonium chrysogenum occurring at intensive synthesis of cephalosporin C has been studied. It is shown, using electron microscopy, that the cell wall of the cells of ATCC 11550 strain (“wild” type) became looser and thicker during growth. The cell wall of the cells of strain 26/8 (hyperautotroph of cephalosporin C) considerably degraded by the end of the stationary phase. Biochemical analysis has shown that these alterations entailed decrease of the proteins’ content covalently or noncovalently linked with the polysaccharides of cell walls of both strains. An increase of sensitivity of cell walls of the strain-hyperautotroph to an activity of lytic enzymes of chitinase, laminarinase, proteinase K, and lyticase preparation has been observed during the growth, but this increase has not been found in the case of “wild” type strain. The obtained results evidence to the structure failure of the cell wall of A. chrysogenum entailing the intensive creation of antibiotic.
Regulatory role of monoamine neurotransmitters in Saccharomyces cerevisiae cells by K. D. Malikina; V. A. Shishov; D. I. Chuvelev; V. S. Kudrin; A. V. Oleskin (pp. 620-625).
Proliferation of Saccharomyces cerevisiae EPF cells on solid maltose-peptone-yeast extract (MPY) medium was stimulated by the addition of monoamine neurotransmitters. Dopamine turned out to be the most efficient among them: it caused ∼8-fold growth stimulation at 1 μM concentration. The dopamine effect was partly mimicked by apomorphine, a dopamine receptor agonist. Serotonin and histamine produced less significant (1.5–2-fold) effects, and norepinephrine virtually failed to stimulate yeast culture growth. These data point to a specific, apparently receptor-dependent mode of action of the tested neurotransmitters on S. cerevisiae cells. Using high performance liquid chromatography, serotonin, catecholamines (dopamine and norepinephrine), catecholamine precursor dioxyphenylamine, and oxidized amine products (homovanilic acid, dihydrophenylacetic acid, and 5-hydroxyindolacetic acid) were established to be accumulated in yeast cells up to (sub)micromolar concentrations without their release into the culture fluid supernatant (CFS). The results obtained suggest that the tested amine neurotransmitters and related compounds do not serve as autoregulators in the yeast population. Nevertheless, they may be involved in the regulation of yeast population development by other ecosystem components.
Purification and characterization of an intracellular β-glucosidase from the protoplast fusant of Aspergillus oryzae and Aspergillus niger by F. -M. Zhu; B. Du; H. -S. Gao; C. -J. Liu; J. Li (pp. 626-632).
Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4,000 with 0.01 mM CaCl2. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular (β-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 5.4 and temperature of 65°C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0–6.6. Na+, K+, Ca2+, Mg2+ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe3+. The enzyme activity was strongly inhibited by glucose, the end product of glucoside hydrolysis. The K m and V max values against salicin as substrate were 0.035 mM and 1.7215 μmol min−1, respectively.
Increase in glucoamylase productivity of Aspergillus awamori strain by combination of radiation mutagenesis and plasmid transformation methods by Yu. P. Vinetsky; A. M. Rozhkova; A. S. Sereda; N. V. Tsurikova; A. K. Nurtaeva; M. V. Semenova; I. N. Zorov; A. P. Sinitsyn (pp. 633-640).
Increase in the expression level of amylolytic genes activator protein encoded by amyR gene was shown to result in enhancement of glucoamylase productivity of A. awamori strain by 30%. However, the same effect equal to 30% increase can be achieved by introduction of extra copies of gla gene encoding glucoamylase. These two effects were not additive, which gave the possibility to suggest an additional limitation in the regulation mechanism of glucoamylase gene expression in A. awamori strain while introducing an additional copies of amyR and gla genes.
Xylanase of the micromycete Rhizopus var. microsporus 595: Preparation, structural and functional characteristics, and application by G. P. Shuvaeva; M. G. Sysoeva (pp. 641-647).
Procedures for the production of endo-1,4-β-xylanase have been developed. An active producer—Rhizopus var. microsporus BKMF-595—has been chosen, and the conditions of surface and submerged cultivation, as well as the composition of the culture medium for this strain, have been optimized to ensure maximum yield of the target enzyme. Activity of xylomicrosporin Px equaled 123 U/g, while the activity of xylomicrosporin Gx equaled 25 U/cm3. Homogeneous enzyme preparations, purified 59.44-fold and 72.6-fold, have been obtained. The dependence of endo-1,4-β-xylanase catalytic activity on temperature and pH of the reaction medium has been studied. The enzyme has been shown to be most stable in the pH range 5.0–6.0 and to be thermostable. Amino acid composition and subunit structure of the enzyme were determined; the molecular masses of the subunits equaled 50 and 56 kDa. Carboxyl groups of glutamic and aspartic acid residues of the active center were experimentally shown to play an important role in catalysis. The potential of this enzyme for beer production has been demonstrated.
Distribution of O-glycosylhydrolases in marine fungi of the Sea of Japan and the Sea of Okhotsk: Characterization of exocellular N-acetyl-β-D-glucosaminidase of the marine fungus Penicillium canescens by Yu. V. Burtseva; V. V. Sova; M. V. Pivkin; S. D. Anastyuk; V. I. Gorbach; T. N. Zvyagintseva (pp. 648-656).
The capacity to produce exocellular enzymes was studied for 92 samples of fungi from various marine habitats in the Sea of Okhotsk (78 strains) and the Sea of Japan (14 strains). Strains producing highly active glycanases and glycosidases were found. Synthesis of O-glycosylhydrolases was stimulated by addition of laminaran to the nutrient medium. Highly purified N-acetyl-β-D-glucosaminidase was isolated from the marine fungus Penicillium canescens. The molecular weight of the enzyme determined by SDS-Na-electrophoresis was 68 kDa. The enzyme displayed maximum activity at pH 4.5 and temperature 45°C. Inactivation half-time of the enzyme at 50°C was 25 min. N-acetyl-β-D-glucosaminidase hydrolyzed both β-glucosaminide and β-galactosaminide bonds and possessed a high transglycosylating activity.